This invention relates to new and improved apparatus and method for the measurement of the sperm density in a specimen and the determination of dosages for artificial insemination.
When properly practiced, artificial insemination (AI) can be a highly successful management technique for improving the reproductive efficiency of various domestic animals.
AI is widely practiced in the cattle breeding industry; is finding increasing use in equine (all breeds except thoroughbred) reproduction; and is being used, at least experimentally in the breeding of canines and other animals.
There are many specific reasons for the practice of AI, most of which ultimately reduce to a matter of economics. For example, an AI program conducted by competent practitioners using proven techniques and equipment can reduce the occurrence of injury to valuable animals and diminish the transmission of certain reproductive system diseases. Additionally, and of major importance, a maximum number of females can be brought into pregnancy in minimum time and the number of progeny from males of recognized genetic superiority can be greatly increased.
Although the detailed procedures and the apparatus used in an AI program vary with the characteristics and anatomy of the various species, the handling, processing and evaluation of semen in the field laboratory is nearly the same for all animals.
It is important that the semen be protected from sunlight and that it be held at nearly constant animal body temperature from the time that it is collected until insemination is accomplished. All apparatus and containers that will come into contact with any semen that is to remain viable must be clean, warmed before use and held at constant temperature wherever possible. In some species, such as the equine, the ejaculate contains a gel-like substance which has no bearing on fertility but does interfere with laboratory evaluation and insemination procedures. The gel is usually removed at the time of collection by passing the ejaculate through an appropriate filter mesh prior to its entry into the holding container. Ideally, semen which has been properly collected from a healthy animal is essentially free of gel and contaminants and consists only of spermatozoa suspended in a clear plasma.
A sample of a uniform mix of the collected semen is drawn for laboratory analysis during which time the bulk of the semen is stored in a properly regulated incubator. Even under the best of storage and handling conditions, various changes and a gradual deterioration in the quality of the semen will occur over a period of time. The rate of deterioration differs as time progresses and tends to be greater in the laboratory sample than in the incubated semen.
The laboratory work, then, must be conducted with great care yet be accomplished as rapidly and efficiently as possible to assure good correlation between the laboratory measurements and stored semen and provide inseminating fluid of the highest possible potency.
The laboratory evaluation of the semen may be quite extensive. However, in routine work a minimal evaluation may be all that is required. A minimal evaluation includes at least the following points:
1. An initial visual and olfactory examination to appraise semen quality and detect the presence of contaminants (dirt, urine, blood, etc.). PA1 2. A measurement of the total volume of ejaculate. PA1 3. An estimate of semen fertility. PA1 4. A determination of the spermatozoa concentration or density (number of sperm cells per unit volume). PA1 5. A calculation of the total number of sperm cells per ejaculate. PA1 6. A determination of the volume of gel-free semen needed for each insemination. PA1 D=Dose or volume of semen in milliliters (ml) to be used for insemination. PA1 N=Total number of viable spermatozoa desired for insemination in millions. PA1 C=Measured concentration or density of sperm in millions of cells per ml. PA1 M=Estimated percentage of sperm that are viable (usually percent forward motility) expressed as a decimal fraction. PA1 a direct digital readout of sperm density to eliminate the need for the use of graphs and charts; PA1 an automated system for optimizing measurement accuracy at low, median and high density counts with the use of only one specified dilution ratio for all measurements; PA1 a "SPECIMEN BELOW RANGE" indicator coupled with a forced zero panel display for densities that are impractically low or below the calibrated range of the instrument; PA1 an insemination dose computer featuring automatic entry of sperm density, digital thumbwheel entry of percent motility and total sperm per insemination, and automatic solution of the dose equation with digital display of the required dose.
Items 1 and 2, sensory evaluation and measurement of volume require no additional comment for purposes of this application.
Item 3, estimation of sperm fertility, is accomplished by a subjective microscopic examination of a standardized dilution of semen to determine the percentage of sperm that are viable. In routine practice, this is done by observing the motility patterns of the spermatozoa and estimating the percentage that move in a typical progressive or forward manner. Other types of movement that have been categorized are circular, vibratory, reverse and flagellating. These movement patterns are thought to be indicative of sperm cells that are unlikely to be capable of achieving ovum fertilization. A more detailed evaluation is sometimes used which includes a study of cell morphology under high power magnification. The extent to which the sample is free of cells having certain physical deformities which are thought to affect fertility may be used to refine the fertility rating.
Item 4, determination of spermatozoa density, may be accomplished by any of the several different methods, four of which are: (1) estimation based upon visual observation of semen turbidity under standardized conditions; (2) visual count using a microscope and hemocytometer; (3) electronic counting and (4) photoelectric techniques in which a measured optical parameter of a standardized sample is correlated to sperm cell density.
Each method has particular advantages and disadvantages. Visual estimation is rapid and inexpensive but yields the poorest accuracy. Hemocytometer counts can be accurate but are tedious and time consuming. Properly performed electronic counting using modern instrumentation probably provides the highest accuracy but the equipment is expensive and the technique seems better suited to laboratory research and to the gathering of calibration data than to practical field use. Photoelectric instrumentation appears to offer the best compromise when factors of accuracy, rapidity of measurement, required operating expertise, cost and equipment portability are all considered and therefore has received a high degree of acceptance.
Item 5, total sperm per ejaculate, is simply the product of ejaculate volume and sperm density. This is generally considered to be the best numerical indication of semen quality.
Calculation of the volume of gel-free semen needed for each insemination, item 6, is done by substituting measured and selected values into a simple equation as follows: EQU D=N/(CM)
Where
The total number of viable sperm cells desired for insemination, N, is the minimum number estimated to yield a high probability of achieving ovum fertilization. The selected quantity depends upon the species of animal, any knowledge of the probable degree of fertility of a particular female (from previous experience), and the expertise and judgement of the AI practitioner. As an example, the selected total number of viable sperm cells for insemination of a mare would normally fall within the range of 100 million to 500 million.
The sperm cell concentration or density, C, and the percent forward motility, M, are both taken from data gathered in the laboratory analysis of the collected semen.
A review of the six steps required for a minimal evaluation of the collected semen shows that most are simple measurements or calculations or are subjective evaluations. Only two items require or lend themselves to the use of sophisticated instrumentation as a means of improving the rapidity and accuracy of semen evaluation. One of the items, estimation of semen fertility, can be enhanced through the use of a high quality phase-contrast type microscope. The evaluation, of course, remains subjective. As alluded to earlier, the second item that can be instrumented is the measurement of spermatozoal density. A photoelectric method for estimating sperm concentration in semen was first reported in 1939 by Comstock and Green for the ram.
Several scientific studies have since been made by other investigators to determine the relationship between the sperm concentration of various domestic animals and such photometric parameters as light scattering (or haze), transmission and absorbance. Both duplicate hemacytometer counts and electronic counting have been used to establish the independent variable.
Before measurement, the semen is diluted by a standardized ratio. Various diluents have been used, most consisting of distilled water with a small percentage of sodium citrate or sodium chloride to balance osmotic pressures on the sperm cells. In addition, some investigators have advised the inclusion of formalin to kill and fix the spermatozoa. The standardized ratio of semen to diluent is initially determined by experiment. It is dependent upon the average semen turbidity of the species and the type of instrument to be used. The dilution ratio is chosen to optimize the spread of readings over the usable range of the selected instrument.
The investigations revealed that no photometric parameter could be considered a truly linear function of sperm concentration over the full gamut of densities that might be encountered. However, for several species absorbance was discovered to be tolerably linear over a rather wide practical range of median concentrations, with slope changes being evident usually toward both extremes of the scale. Acceptable accuracy could often be achieved by assuming a linear relationship for a practical range of values followed by smoothing the measurement deviations by using a linear least squares regression analysis to derive a best-fit calibration equation.
A technique that has been devised to provide improved accuracy where needed is to specify two (or more) dilution ratios with corresponding linear regression equations. In effect, this forms multiple overlapping ranges with a portion of each range falling over the most linear segment of the instrument characteristic. A subjective evaluation of semen turbidity is then used to select the dilution ratio-equation set that will optimize the measurement accuracy.
Another research finding was that the dominant radiation wavelength used for absorbance measurement is not a critical factor, at least over the range of the visible spectrum (380 nm to 760 nm). Absorbance simply decreases in a smooth and fairly gradual manner as wavelength is increased. This indicates that any wavelength of convenience (or even a wide band of wavelengths) may be used providing that measurements are made under the same conditions that were established for calibration of the instrument.
For a number of years, objective measurements of sperm concentration have been successfully accomplished in the field using specially calibrated general purpose laboratory spectrophotometers. Most of the instruments in use are analog types which were primarily designed for measurement of percent transmission of light through test samples at a selected (narrow band) wavelength. The more modern of these instruments include an absorbance scale which is logarithmically related to percent transmission (absorbance equals 2 minus log.sub.10 of the percent transmission). Because a linear transmission scale can usually be interpolated and read more accurately than a non-linear absorbance scale, many of the operational procedures in use are based upon measurement of transmission. Conversion to absorbance is accomplished mathematically by the operator or, more conveniently, may be part of a computerized program if the sperm density value is taken from computer-derived graphs or charts showing transmission as the independent variable.
An investigation of prior instruments was conducted, but no existing photometer was found to have overall characteristics that would make it particularly suited for use in the determination of sperm density. Accordingly it is an object of the present invention to provide a photometer and associated components which will provide an objective measurement of sperm concentration and which can be used in the field to determine dosages. An additional object is to provide apparatus and method which will eliminate or minimize recognized deficiencies in existing systems and to provide improved accuracy and operating convenience.
It is an object of the invention to provide an instrument which functions on the basis of a direct absorbance measurement to eliminate any requirement for mathematical conversion from transmission and to allow standardization of the specimen cuvette with a single adjustment. Transmission measurements require separate adjustments for zero and full scale.
Further objects of the invention include:
It is an additional object of the invention to provide such an instrument having high reliability and one which will operate with infrequent calibration and function within specifications over an ambient temperature range of 20.degree. F. to 110.degree. F., and one in which the calibration procedure is straightforward with all adjustments being independent and non-iterative.
Other objects, features and results will more fully appear in the course of the following description and claims.